Dependence of hyperpolarisation-activated cyclic nucleotide-gated channel activity on basal cyclic adenosine monophosphate production in spontaneously firing GH3 cells

The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca2+ signalling, and prolactin secretion in GH(3) immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below - 60 mV elicited a slowly activating voltage-dependent inward current (I-h) in the majority of tested cells, with a half-maximal activation voltage of -89.9 +/- 4.2 mV and with a time constant of 1.4 +/- 0.2 s at -120 mV. The bath application of 1 mM Cs+, a commonly used inorganic blocker of I-h, and 100 mu M ZD7288, a specific organic blocker of I-h, inhibited I-h by 90 +/- 4.1% and 84.3 +/- 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect I-h. Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of I-h by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs+ had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and I-h-independent effects on spontaneous electrical activity, Ca2+ signalling, and prolactin release. These results indicate that HCN channels in GH(3) cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials.